A novel treatment to enhance survival for end stage triple negative breast cancer using repurposed veterinary anthelmintics combined with gut­supporting/immune enhancing molecules.

Patients with end­stage metastatic disease have limited treatment options and those diagnosed with triple negative breast cancer (Her2, Estrogen receptor, Progesterone receptor) have a poor prognosis. Using a triple negative mammary tumor model selected for brain metastasis (4T1Br4) in the mouse, treatment options that may increase survival when therapeutics are applied at post­metastasis were assessed. Anti­parasitic benzimidazoles (BZs) destabilize microtubules, inhibit metabolic pathways, reduce cell proliferation, and induce apoptosis in tumor cells. Co­administration of two BZs was selected, oxfendazole (OFZ) and parbendazole (PBZ), shown to overcome resistance development in anthelmintic effects by imposing metabolic delay to assess if multiple BZ approach is also suitable to enhance anticancer effects. It has been previously reported that treatment of mammary tumor­bearing mice at an early stage with chitin microparticles (CMPs) decreased tumor growth and metastases by enhancing both innate M1 macrophage and TH1 adaptive immune response. Oral administration of CMPs was previously revealed to affect the gut in intestinal inflammation. A combination BZ (OFZ/PBZ) and CMP treatment was tested to target tumor development and metastasis and effects were compared in response to monotherapies of the same compounds or to untreated mice. The results demonstrated increased survival, decreased tumor cell proliferation, decreased metastasis in lungs and brain, increased levels of fecal SCFAs butyric, acetic, propionic and valeric acids with increased butyric and propionic acid levels in brain biopsies in combination treated compared with untreated mice. At the primary tumor, SCFA receptor FFAR2 expression was increased in combination treatment compared with untreated mice, suggestive of a non­invasive cancer phenotype. The superior cytotoxic effects of OFZ/PBZ were confirmed as opposed to single treatment with OFZ or PBZ using 3D spheroids generated from a human breast cancer cell line, MDA­MB­468. These data are compelling for treatment option possibility even at late stages of metastasized breast cancer.

Inter-individual differences in immune profiles of outbred rats screened for an emotional reactivity phenotype.

Inter-individual differences in emotional reactivity predict susceptibility versus resilience to mood pathology. Using experimentally-naïve outbred rats that vary in locomotor reactivity to the mild stress of an inescapable novel environment [i.e., top and bottom 1/3rd of the population identified as high responders (HR) and low responders (LR) respectively], we determined baseline variations in immune functions. Innate and adaptive immune responses vary basally in LRHR rats, namely a shift towards TH1 in LRs and TH2 in HRs was observed. These inter-individual variations in immune profiles in LRHRs could have significant implications in mood alterations and immune reactivity to microbes and cancer.

Semaphorins in Angiogenesis and Autoimmune Diseases: Therapeutic Targets?

The axonal guidance molecules, semaphorins, have been described to function both physiologically and pathologically outside of the nervous system. In this review, we focus on the vertebrate semaphorins found in classes 3 through 7 and their roles in vascular development and autoimmune diseases. Recent studies indicate that while some of these vertebrate semaphorins promote angiogenesis, others have an angiostatic function. Since some semaphorins are also expressed by different immune cells and are known to modulate immune responses, they have been implicated in autoimmune disorders such as multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus and systemic sclerosis. We conclude this review by addressing strategies targeting semaphorins as potential therapeutic agents for angiogenesis and autoimmune diseases.

YKL-40/CHI3L1 drives inflammation on the road of tumor progression.

Inflammation plays a vital role at different stages of tumor progression. The development of tumors is affected by inflammatory mediators produced by the tumor and the host. YKL-40/chitinase-3-like-1 protein is often up-regulated in inflammation-associated diseases. With the use of chronic inflammatory disease systems, we describe the role of YKL-40/chitinase-3-like-1 protein in enhancing the inflammatory response and its implications in tumorigenesis. We also discuss how pre-existing inflammation enhances tumor growth and metastasis. In this mini-review, we highlight the effect of YKL-40/chitinase-3-like-1 protein-associated inflammation in promoting tumor progression.

Allergen induced pulmonary inflammation enhances mammary tumor growth and metastasis: Role of CHI3L1.

Metastasis is the primary cause of mortality in women with breast cancer. Metastasis to the lungs is greater in patients with pulmonary inflammatory illnesses. It is unknown how pre-existing pulmonary inflammation affects mammary tumor progression. We developed a novel breast cancer model in which pulmonary inflammation is induced in mice prior to tumor cell implantation. In the present study, we determined how pre-existing allergen-induced inflammation changes the pulmonary microenvironment to exacerbate tumor metastasis. We showed that pre-existing pulmonary inflammation in mammary tumor bearers is associated with: 1) an increase in growth of the primary tumor and metastasis; 2) an increase in the expression of a glycoprotein known as CHI3L1; and 3) increase in the levels of myeloid populations in their lungs. We also showed that myeloid derived cells from the lungs of allergic tumor bearers produce higher amounts of CHI3L1 than the saline controls. We previously showed that CHI3L1 induces the expression of proinflammatory and protumorigenic molecules. In this study, we show that CHI3L1 knockout tumor bearers with pre-existing allergic pulmonary inflammation had decreased levels of myeloid-derived cells, decreased levels of proinflammatory mediators, and a significant reduction in tumor volume and metastasis compared with the wild-type controls. Pre-existing inflammation and CHI3L1 might be driving the establishment of a premetastatic milieu in the lungs and aiding in the support of metastatic foci. Understanding the role of allergen-induced CHI3L1 and inflammation in tumor bearers and its effects on the pulmonary microenvironment could result in targeted therapies for breast cancer.

Semaphorin7A promotes tumor growth and exerts a pro-angiogenic effect in macrophages of mammary tumor-bearing mice.

Semaphorins are a large family of molecules involved in axonal guidance during the development of the nervous system and have been recently shown to have both angiogenic and anti-angiogenic properties. Specifically, semaphorin 7A (SEMA7A) has been reported to have a chemotactic activity in neurogenesis and to be an immune modulator through α1ß1integrins. SEMA7A has been shown to promote monocyte chemotaxis and induce them to produce proinflammatory mediators. In this study we explored the role of SEMA7A in a murine model of breast cancer. We show that SEMA7A is highly expressed by DA-3 murine mammary tumor cells in comparison to normal mammary cells (EpH4), and that peritoneal elicited macrophages from mammary tumor-bearing mice also express SEMA7A at higher levels compared to those derived from normal mice. We also show that murine macrophages treated with recombinant murine SEMA7A significantly increased their expression of proangiogenic molecule CXCL2/MIP-2. Gene silencing of SEMA7A in peritoneal elicited macrophages from DA-3 tumor-bearing mice resulted in decreased CXCL2/MIP-2 expression. Mice implanted with SEMA7A silenced tumor cells showed decreased angiogenesis in the tumors compared to the wild type tumors. Furthermore, peritoneal elicited macrophages from mice bearing SEMA7A-silenced tumors produce significantly (p < 0.01) lower levels of angiogenic proteins, such as CXCL2/MIP-2, CXCL1, and MMP-9, compared to those from control DA-3 mammary tumors. We postulate that SEMA7A in mammary carcinomas may skew monocytes into a pro-tumorigenic phenotype to support tumor growth. SEMA7A could prove to be valuable in establishing new research avenues toward unraveling important tumor-host immune interactions in breast cancer patients.

CHI3L1 plays a role in cancer through enhanced production of pro-inflammatory/pro-tumorigenic and angiogenic factors.

Elevated serum levels of a glycoprotein known as chitinase-3-like protein 1 (CHI3L1) have been correlated with poor prognosis and shorter survival of patients with cancer and inflammatory diseases. The biological and physiological functions of CHI3L1 in cancer have not yet been completely elucidated. In this review, we describe the role of CHI3L1 in inducing pro-inflammatory/pro-tumorigenic and angiogenic factors that could promote tumor growth and metastasis.

Semaphorin7A: branching beyond axonal guidance and into immunity.

Semaphorins are a family of proteins that were originally described for their role in axonal guidance. Studies now show that semaphorins encompass many physiological functions outside of the nervous system, including immune responses. Semaphorin7A (SEMA7A) belongs to the "immune" semaphorin group and has been shown to play a crucial role in regulating immune responses. In this review, we discuss the structure and function of SEMA7A as well as its role in innate and adaptive immunity [corrected].We further describe SEMA7A's involvement in inflammatory disease and its emergent role in cancer.

Tumor microenvironment profoundly modifies functional status of macrophages: peritoneal and tumor-associated macrophages are two very different subpopulations.

Macrophages are key players in the inflammatory response. In this study, we tested the hypothesis that although all macrophage subpopulations in tumor hosts are affected by the disease, it is the close proximity to the tumor that induces major alterations in these cells. We compared tumor-associated macrophages (TAMs) with peritoneal macrophages from mice bearing D1-DMBA-3 mammary tumors (T-PEMs). Our results show that TAMs downregulate IL-12p70 but upregulate IL-12p40, IL-23, IL-6 and IL-10. Some NFκB and C/EBP transcription factors family members are decreased in TAMs; however NFκBp50 homodimers, STAT1/pSTAT1 and STAT3/pSTAT3 are overexpressed. Furthermore, while TAMs block T-cell proliferation and are more prone to apoptosis compared to T-PEMs, both types of macrophages have an impaired phagocytic capacity. Moreover, TAMs constitutively express iNOS and produce nitric oxide but do not express arginase and are Gr-1(high) and CD11b(low). Collectively, our analysis of two spatially distinct macrophage subpopulations in tumor-bearing mice revealed that the tumor modulates them differently into two molecularly and functionally dissimilar macrophage subpopulations.

Exploring the role of CHI3L1 in "pre-metastatic" lungs of mammary tumor-bearing mice.

Elevated levels of chitinase-3-like-1 (CHI3L1) are associated with poor prognosis, shorter recurrence-free intervals and low survival in breast cancer patients. Breast cancer often metastasizes to the lung. We hypothesized that molecules expressed in the "pre-metastatic" lung microenvironment could support the newly immigrant tumor cells by providing growth and angiogenic factors. Macrophages are known to play an important role in tumor growth by releasing pro-angiogenic molecules. Using mouse mammary tumor models, we have previously shown that during neoplastic progression both the mammary tumor cells and splenic macrophages from tumor-bearing mice express higher levels of CHI3L1 compared to normal control mice. However, the role of CHI3L1 in inducing angiogenesis by macrophages at the pulmonary microenvironment to support newly arriving breast cancer cells is not yet known. In this study, we determined the expression of CHI3L1 in bronchoalveolar lavage macrophages and interstitial macrophages in regulating angiogenesis that could support the growth of newly immigrant mammary tumor cells into the lung. Here we show that in vitro treatment of pulmonary macrophages with recombinant murine CHI3L1 resulted in enhanced expression of pro-angiogenic molecules including CCL2, CXCL2, and MMP-9. We and others have previously shown that inhibition of CHI3L1 decreases the production of angiogenic molecules. In this study, we explored if in vivo administration of chitin microparticles has an effect on the expression of CHI3L1 and pro-angiogenic molecules in the lungs of mammary tumor-bearing mice. We show that treatment with chitin microparticles decreases the expression of CHI3L1 and pro-angiogenic molecules in the "metastatic" lung. These studies suggest that targeting CHI3L1 may serve as a potential therapeutic agent to inhibit angiogenesis and thus possibly tumor growth and metastasis.

Induction of proinflammatory mediators by CHI3L1 is reduced by chitin treatment: decreased tumor metastasis in a breast cancer model.

Disseminated metastasis accounts for over 90% of breast cancer deaths. Recently, elevated serum levels of a glycoprotein known as chitinase-3 like-protein-1 (CHI3L1) has been correlated with poor prognosis and shorter survival of patients with metastatic breast cancer. In this study, we show that there are increased levels of CHI3L1 in plasma of tumor-bearing mice and that both tumor cells and immune cells express and secrete CHI3L1. However, the biological and physiological functions of CHI3L1 are still unclear. We demonstrate that while CHI3L1 has an inhibitory role in the expression of interferon-gamma (IFN-γ), CHI3L1 up-regulates pro-inflammatory mediators, C-chemokine ligand 2 (CCL2), chemokine CX motif ligand 2 (CXCL2) and matrix metalloproteinase-9 (MMP-9) all of which contribute to tumor growth and metastasis. We found that in vitro inhibition of CHI3L1 by siRNA suppressed the production of CCL2, CXCL2 and MMP-9 by macrophages. In vivo treatment of mammary tumor-bearing mice with chitin (ß-(1-4)-poly-N-acetyl D-glucosamine), a TH(1) adjuvant and a ligand for CHI3L1, promoted immune effector functions with increased production of IFN-γ and decreased CCL2, CXCL2 and MMP-9 expression. In vivo administration of chitin to mammary tumor-bearing mice significantly decreased lung metastasis. These studies show that CHI3L1 plays a role in tumor progression and that chitin can inhibit the pleiotropic effects of CHI3L1 giving support to the idea that CHI3L1 is a useful therapeutic target for treatment of breast cancer.

Decreased accumulation of immune regulatory cells is correlated to the antitumor effect of IFN-γ overexpression in the tumor.

During mammary tumorigenesis, there is a profound tumor-induced immunosuppression and a progressive thymic atrophy associated with tumor development. IFN-γ has been shown to be effective in enhancing antitumor responses in several tumor models, however, how IFN-γ exerts its anti-tumor effect is largely controversial. In the present study we have used a mammary tumor model to investigate whether the levels of IFN-γ have an important role in the tumor-induced immuno-suppression as well as in the pathogenesis of the thymic atrophy. We evaluated this possibility using DA-3 cells transfected to express IFN-γ (DA-3/IFN-γ), a system that provides constant, local production of IFN-γ within the tumor microenvironment. Overexpression of IFN-γ in the mammary tumor results in a marked delay of tumor growth, a reduction in regulatory T cells and myeloid-derived suppressor cells accumulation mostly due to down-regulation of chemokines implicated in the recruitment of immune regulatory cells, and a blockage in the tumor-associated thymus atrophy. Collectively, our data suggest that the replacement of the faulty levels of IFN-γ in the tumor results in a diminution of the tumor-induced immune suppression caused by the mammary tumor development.

Expression of the inflammatory chemokines CCL2, CCL5 and CXCL2 and the receptors CCR1-3 and CXCR2 in T lymphocytes from mammary tumor-bearing mice.

Chemokines and their receptors have been studied in several solid tumor models as mediators of inflammation. In turn, inflammation has been implicated in the promotion and progression of tumors, and as such, chemokines have been proposed as novel molecular targets for chemotherapy. While the expression of these molecules has been described in tumor cells, endothelial cells, macrophages and neutrophils, less attention has been paid to the expression profile of these molecules by T lymphocytes in the periphery or infiltrating the tumor. Using the D1-DMBA-3 murine mammary adenocarcinoma model, we aimed to better characterize the differential expression of chemokines and/or their receptors in the host and in the tumor microenvironment, and specifically, in the T cells of tumor-bearing mice compared to normal control animals. We found that T lymphocytes from tumor-bearing mice express the pro-inflammatory chemokines, CCL2, CCL5 and CXCL2, as well as the chemokine receptors, CCR1, CCR2, CCR3 and CXCR2.

Tumor-induced thymic atrophy: alteration in interferons and Jak/Stats signaling pathways.

The thymus is the major site of T cell differentiation and a key organ of the immune system. Thym atrophy has been observed in several model systems including aging, and tumor development. Previous results from our laboratory have reported that the thymic atrophy seen in mammary tumor bearers is associated with a severe depletion of CD4+CD8+ double positive immature cells and changes in the levels of cytokines expressed in the thymus microenvironment. Cytokines regulate numerous aspects of hematopoiesis via activation of the Jak/Stat pathways. In the present study we have used our mammary tumor model to investigate whether changes in the levels of cytokines in the thymus could affect the normal expression of the aforementioned pathways. RNA and protein analysis revealed an overexpression of the different members of interferons, a downregulation of most of the Jak/Stat pathways, and an increased expression of several suppressors of cytokine signaling (SOSC) in the thymuses of tumor bearers. Together, our data suggest that the impaired Jak/Stat signaling pathways observed in the whole thymus of tumor-bearing mice could be contributing to the abnormal T cell development and apoptosis observed during the tumor-induced thymic atrophy.

Decreased levels of both Stat1 and Stat3 in T lymphocytes from mice bearing mammary tumors.

BACKGROUND: Lymphocytes from tumor-bearing animals have been shown to lack antitumor function. The objective of this study was to investigate the status of the signal transducers, Stat1 and Stat3, in T lymphocytes of animals bearing D1-DMBA-3 mammary tumors and to elucidate if any alterations in these signal transducers can be explained by the presence of tumor-derived factors and correlated with the lack of antitumor function in these cells. MATERIALS AND METHODS: T Lymphocytes from spleens of normal and tumor-bearing mice were purified and assayed for the presence of Stat1 and Stat3 by Western blot analysis. RESULTS: It was found that levels of both Stat1 and Stat3 were reduced in T lymphocytes of tumor-bearers not only in their active, phosphorylated form but in total protein levels. CONCLUSION: These findings indicate that during mammary tumor progression, alteration of various transcription factors may contribute to the down-regulation of immune function.

Identification of a subpopulation of macrophages in mammary tumor-bearing mice that are neither M1 nor M2 and are less differentiated.

Systemic and local immune deficiency is associated with cancer, and the role of M2 tumor-associated macrophages in this phenomenon is well recognized. However, the immune status of macrophages from peripheral compartments in tumor hosts is unclear. Peritoneal macrophages (PEM) are derived from circulating monocytes and recruited to the peritoneal cavity where they differentiate into macrophages. We have previously shown that PEMs from mice bearing D1-DMBA-3 mammary tumors (T-PEM) are deficient in inflammatory functions and that this impairment is associated with diminished expression of transcription factors nuclear factor kappaB and CAAT/enhancer-binding protein. We now provide evidence that T-PEMs display neither M1 nor M2 phenotypes, yet exhibit deficiencies in the expression of several inflammatory cytokines and various proinflammatory signaling pathways. Moreover, due to nuclear factor kappaB down-regulation, increased apoptosis was observed in T-PEMs. We report for the first time that macrophage depletion is associated with increased macrophage progenitors in bone marrow. Furthermore, T-PEMs have a lower expression of macrophage differentiation markers F4/80, CD68, CD115, and CD11b, whereas Gr-1 is up-regulated. Our results suggest that T-PEMs are less differentiated and represent a newly derived population from blood monocytes. Lastly, we show that transforming growth factor-beta and prostaglandin E(2), two immunosuppressive tumor-derived factors, may be involved in this phenomenon.

Molecular events involved in the increased expression of matrix metalloproteinase-9 by T lymphocytes of mammary tumor-bearing mice.

Matrix metalloproteinases (MMPs) are a family of extracellular proteinases whose contributions to cancer progression have been studied because of their matrix-degrading abilities and elevated expression in advanced stage tumors. Recent findings suggest a role for MMPs during the multiple stages of tumor progression including establishment and growth, migration, invasion, metastasis, and angiogenesis. MMP-9 regulation at the molecular level can be studied by measuring the effect(s) of a variety of physiological and pharmacological agents on cells. Multiple signaling molecules such as protein kinase C, pertussis toxin-sensitive guanine nucleotide-binding protein G, and protein tyrosine kinases are known to mediate the secretion of MMPs in cell lines. We previously reported an upregulation of MMP-9 in T cells of mammary tumor-bearing mice. In this study, pharmacologic inhibitors were used to dissect the signaling pathways involved in the upregulation of MMP-9 in the splenic T cells of normal and mammary tumor-bearing mice. Staurosporine, a protein kinase inhibitor, stimulated MMP-9 secretion by normal T lymphocytes, while the constitutively high levels of MMP-9 produced by tumor bearers' T cells were decreased by Genistein, a specific tyrosine kinase inhibitor, and Rottlerin, a PKC inhibitor. Using a NF-kappaB specific probe to the murine MMP-9 promoter, electromobility shift assays of nuclear proteins from normal and tumor bearers' splenic T cells revealed a pattern of higher intensity bands from the tumor bearers' nuclear extracts, indicating a greater amount of these transcription factors bound to the recognition motif. When mammary tumor bearers' T cells were cultured with the NF-kappaB inhibitors, N-p-Tosyl-L-lysine chloromethyl ketone hydrochloride and Bay 11-7082, there was a subsequent decreased production of MMP-9. These results suggest that the tumor burden may be activating various signaling pathways within splenic T lymphocytes to upregulate MMP-9 expression.

GM-CSF up-regulates the expression of CCL2 by T lymphocytes in mammary tumor-bearing mice.

MCP-1/CCL2 (monocyte chemoattractant protein-1/CC chemokine ligand 2) is a beta or CC chemokine that is expressed by a variety of cell types, including fibroblasts, endothelial, smooth muscle, and glial cells. In addition, cells involved in immunity, such as monocytes/macrophages, neutrophils, and eosinophils have also been shown to express this chemoattractant. Using a murine model of the D1-DMBA-3 mammary adenocarcinoma, we demonstrated the unique production of CCL2 by splenic T lymphocytes from tumor-bearing animals. Because this tumor produces GM-CSF, and this factor is also up-regulated in the B lymphocytes of tumor-bearing mice, we looked at the ability of GM-CSF to induce CCL2 production by T cells. Treatment of normal and tumor bearers' T cells with GM-CSF resulted in an increased secretion of this chemokine. This up-regulation was seen with or without stimulation by Concanavalin A, although these treatments were additive in their effects. The induction of CCL2 was studied at the molecular level by analyzing the effect(s) of a variety of physiological and pharmacological agents on cultured T cells. These results suggest that the tumor-derived factor GM-CSF activates various signaling pathways within splenic T cells to up-regulate CCL2 expression.